The goal of this research is to determine the mechanism by which Naegleria fowleri induces cytopathic effects in nerve cells in culture. The elucidation of the mechanism by which these effector cells cause target cell death would provide basic information as to the nature of effector-target cell interactions. Furthermore, recognition of the basic character of these cell-cell interactions would provide a basis for understanding the disease process caused by the pathogenic strains of N. fowleri, which induce primary amebic meningoencephalitis. In the first phase of this study, it is proposed to define whether pathogenic strains and low pathogenic strains of Naegleria induce cytopathology by a common mechanism. N. fowleri amebae exhibit a unique structure, the "food-cup" or amebostome. It is proposed to determine whether "food-cup" expression correlates with target cell destruction. Metabolic inhibitors such as cycloheximide and dinitrophenol, and microtubular and microfilament function inhibitors will be employed to study attachment and target cell killing. Scanning electron microscopy and a 51 Cr-release assay will be employed to monitor attachment and cytolysis, respectively. In the second phase, it is proposed to define which factors elicited by nerve cells and other mammalian cells serve as chemoattractants of Naegleria. Chemotaxis assays will include the use of Boyden chambers. In the third phase, the nature of Naegleria cytotoxin will be defined biologically, biochemically and immunologically. Sequential ammonium sulfate precipitation, gel filtration, and preparative isoelectrofocusing will be employed to purify the cytotoxic factor from crude lysates of N. fowleri. Characterization of the cytotoxic factor(s) will include determination of molecular weight, isoelectric points, reaction with reducing and alkylating agents, and inhibitors of proteases nd phospholipases. In the fourth phase, antiserum to the cytotoxic material will be prepred in rabbits. The antiserum will be used for determining the location of the cytotoxic factor on or within the amebae and for determining the location of the cytotoxin on or within the target nerve cell. We propose to study the mechanism of action of the cytotoxic factor on nerve cells by utilizing electron microscopy and radioisotope labeling techniques. We will label target cells with 86 Rb, 35 S-methionine, and 3 H-thymidine to determine if ion efflux occurs prior to cytoplasmic protein or nuclear content extrusion. In conducting these studies, the production of cytotoxins will be correlated to amebostome expression.